Cuiying Xiao

The shorter zinc finger protein ZNF230 gene message is transcribed in fertile male testes and may be related to human spermatogenesis

The zinc finger gene family represents one of the largest in the mammalian genome, with several of these genes reported to be involved in spermatogenesis. A newly discovered gene has been identified that is expressed abundantly in the testicular tissue of fertile men as determined by mRNA differential display. The gene encodes a C(3)HC(4)-type zinc finger protein motif (ring finger motif) consistent with a role in pre-meiotic or post-meiotic sperm development. The gene was named ZNF230 and mapped to the short arm of chromosome 11 (11p15). ZNF230 has two transcripts, of 1 kb and 4.4 kb in length. The shorter 1 kb transcript was only detected in testicular tissue whereas the longer 4.4 kb transcript was not detected in testis but was found in several other tissues. The lack of detectable ZNF230 expression in azoospermic patients by reverse transcriptase-mediated PCR analysis is interpreted to mean that this gene is involved in maintaining normal human male fertility.

Isolation, characterization, and mapping of a novel human KRAB zinc finger protein encoding gene ZNF463.

A novel human KRAB (Krüppel associated box) type zinc finger protein encoding gene, ZNF463, was obtained by mRNA differential display and RACE. It consists of 1904 nucleotides and encodes a protein of 463 amino acids with an amino-terminal KRAB domain and 12 carboxy-terminal C2H2 zinc finger units. The gene is mapped to chromosome 19q13.3 approximately 4 by FISH. As from Northern blot analysis ZNF463 is only expressed in testis, RT-PCR indicates that ZNF463 is expressed more highly in normal fertile adults than in fetus and azoospermic patients suggesting that it may play a role in human spermatogenesis.

8302A/C and (TTA)n polymorphisms in the HMG-CoA reductase gene may be associated with some plasma lipid metabolic phenotypes in patients with coronary heart disease.

HMG-CoA reductase (HMGCR) is a rate-limiting enzyme that participates in cholesterol metabolism. Here we analyzed the 8302A/C and the (TTA)n polymorphisms in the HMGCR gene in 169 Chinese patients with coronary heart disease (CHD) and 161 age-matched controls. Results indicated that the levels of plasma VLDL and TG in patients with the AA genotype of the 8302A/C locus were significantly higher than in patients with other genotypes (P<0.05). In addition, the frequency of allele A4 of the (TTA)n locus was higher (P<0.05) and the frequency of allele A5 was lower (P=0.002) in CHD patients than in the controls. This suggests that both polymorphisms in the HMGCR gene may be associated with lipid and lipoprotein abnormalities in CHD in the Chinese.

A single nucleotide deletion of 293delT in SEDL gene causing spondyloepiphyseal dysplasia tarda in a four-generation Chinese family.

Spondyloepiphyseal Dysplasia Tarda (SEDT; MIM 313400) is a rare genetically heterogeneous disorder of vertebral and epiphyseal growth resulting in disproportionally short-trunked short stature, barrel-shaped chest, and dysplasia of the large joints. It is caused by the mutations of SEDL gene. The distinctive radiological signs and the X-linked mode of inheritance make it easy to diagnose. Here a four-generation Chinese SEDT family has been analyzed and the disease-causing mutation has been found. After polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis and DNA sequencing, a previously unreported deletion of T in exon 5 of SEDL gene (i.e. 293delT) was observed and seven individuals in the family carried the mutation. It results in frameshift and a putative truncated protein with the 97 N-terminal amino acids, and 9 changed amino acids. Therefore, loss of function of the gene could be predicted. However, this mutation has not been detected in 50 age and sex matched unrelated controls.

Uniform Terminus PCR: Amplification of Minute Unknown DNA Fragments

Most published methods for obtaining unknown DNA fragments involve ligation-mediated polymerase chain reaction (PCR) that allows the amplification of unknown sequences flanking short known fragments of DNA. The methods require using a single specific primer (or a pair of nested primers) and a nonspecific primer complimentary to either plasmid sequence or a linker DNA duplex ligated to blunt or sticky DNA ends (1–5). Here, we report an easily implemented modification for amplifying unknown DNA fragments that is named uniform terminus PCR (UT-PCR). We have successfully used this method to amplify and clone DNA fragments from different genes. As an example, we describe the amplification of the (CTG)n repeats of the 3′-untranslated region (3′-UTR) in human myotonin protein kinase (MT-PK) gene. This method has also been used to analyze the termini of PCR products amplified by using Taq DNA polymerase. The key point of the modification is to synthesize a linker with complete or incomplete palindromic structure as its stem part and three random nucleotides at its 3′ end (PPL in our experiment), and an amplification primer containing only the sequence of the stem part of the linker (PP in our paper). The 5′ region of the PPL has been previously phosphorylated to facilitate its ligation to the single-stranded (ss)DNA to be amplified. The last nucleotide in its 3′ end is a ddNTP, which avoids the linker/linker ligation; and during PCR cycling, the extension from the linker residues left from the ligation step is blocked. The 3′ protruding end of the linker improves ligation efficiency greatly and permits its application to both double-stranded (ds)DNA and ssDNA. The palindromic structure of the linker assures the 3′-NNN end anneals to the 3′ end, rather than other sites of the template DNA, which is important for obtaining full-length PCR products of interest. During the amplification, the palindromic structure in the primer leads to the annealing of most of the primers, leaving limited amount of free primer molecules. The latter can anneal more specifically to the stem part of the linker ligated to the 3′ end of ssDNA, therefore greatly raising the specificity of UT-PCR. The three restriction sites in the primer facilitate the cloning after amplification. The PPL molecules are incubated at 65°C for 10 min and slowly cooled down to room temperature to form an almost perfect double-stranded structure, leaving the three random nucleotides free. Figure 1 shows the annealing reaction. It is the 3′-NNN in the annealed double-stranded PPL that can anneal to any ssDNA, irrespective of the DNA fragments being blunt or sticky, and their sequence being known or not, which makes the subsequent amplification possible and effective. Thus, only one primer has to be made, and this primer is used for any UT-PCR application. First, the DNA fragments to be amplified were heat-denatured to generate ssDNA. Then, the dsPPL was annealed and ligated to the denatured ssDNA. Third, the ligated DNAs were denatured in the presence of PP. Finally, the PP served as an amplification primer, and the PCR was carried out. Briefly, about 30 ng (ca. 0.3–0.5 pmol) of heatdenatured PCR products of (CTG)n repeats, with flanking sequences in MTPK gene amplified from genomic DNA, were mixed with 2 U of T4 DNA ligase in a 20–30-μL reaction volume containing 1× Ligation Buffer (Life